Process for manufacture of a modified collagen-induced platelet aggregation inhibitor pallidipin

ABSTRACT

The invention provides a process of manufacture of a recombinant protein called Asp-Pallidipin. Asp-Pallidipin inhibits the collagen-induced platelet aggregation of mammalian platelets. The Asp-Pallidipin comprises 
     (i) a protein (Pallidipin) selected from the group of Pallidipin proteins, and 
     (ii) the amino acid aspartic acid, 
     wherein the aspartic acid is connected by a peptide bond with the N-terminal end of Pallidipin. The process comprises the steps 
     aa) transfecting at least one bacterium with an appropriate vector, wherein the vector comprises: 
     (i) a DNA or cDNA coding for the recombinant Asp-Pallidipin 
     (ii) a suitable signal peptide sequence which signal sequence is cleaved so that the amino acid aspartic acid is in the position +1 of the amino acid sequence seen from the position of the Pallidipin and 
     (iii) a suitable promoter; 
     bb) expressing the preprotein comprising Asp-Pallidipin and the signal sequence; 
     cc) transporting the Asp-Pallidipin from the cytoplasm of the bacterium to the periplasm, 
     cleavage of the preprotein by at least one protease during the transport, producing the Asp-Pallidipin, 
     dd) isolating the Asp-Pallidipin by extracting the periplasm, and 
     ee) purifying the Asp-Pallidipin. 
     The protein is used as a medicament for inhibiting of collagen-induced human platelet aggregation or of cancer with metastatic tumor cells.

The invention refers to a process of manufacture of a modifiedcollagen-induced platelet aggregation inhibitor, called Pallidipin.Further the invention comprises the substance Pallidipin which ismodified.

Collagen is the most potent inducer known of human platelet aggregation.For instance, upon injury of the vessel wall and exposure to collagen,blood platelets rapidly adhere and become activated. (H. R. Baumgartner(1977) Thromb. Haemostas. 37:1-16; J. Hawiger (1987) Human Pathol.18:111-122.)

Collagen-induced platelet aggregation of human platelets thus representsa risk factor for patients undergoing blood vessel-affecting procedures,e.g., angioplasty or sepsis, for those suffering myocardial infarction,for those recovering from treatment for myocardial infarction, interalia.

In some cases it is necessary to inhibit collagen-induced plateletaggregation. Some compounds are known to inhibit such aggregation. Forexample, synthetic oligopeptides inhibit collagen-induced plateletaggregation by binding to the platelets. See, e.g., Bevers et al. (1985)"Collagen Derived Octapeptide Inhibits Platelet Procoagulant ActivityInduced by the Combined Action of Collagen and Thrombin", ThrombosisResearch, 37:365-370; Karniguian et al. (1983) "Effect of a CollagenDerived Octapeptide on Different Steps of the Platelet/CollagenInter-action", Thrombosis Research 32:593-604; Caen et al. (1981)"Oligo-peptides with specific inhibiting properties of collagen-inducedaggregation, process for preparing the same and pharmaceuticalcompositions containing them"; and EPA 0 040 149.

Another source of collagen-induced platelet aggregation inhibitor is aninhibitor identified in a snake venom having an unknown structure. SeeSmith et al., "Identification of 50 kDalton snake venom proteins whichspecifically inhibit platelet adhesion to collagen." (1991) FEBS283:307-310.

A third such inhibitor, from the saliva of a medicinal leech, isdescribed by Munro et al. (1991) "Calin--a platelet adhesion inhibitorfrom the saliva of the medicinal leech", Blood Coagulation andFibrinolysis 2:179-184. The publication of the European patentapplication EP 0 480 651 (Merck & Co. Inc. published 15 Apr. 1992)describes a protein having a molecular weight of about 16 kDalton (kD)and a capacity to inhibit collagen-induced aggregation of humanplatelets which protein is derived from the salivary gland of the leechHaemaenteria officinalis. LAPP is 16 kDa protein from the leechHaemaenteria officinalis described in Connolly et al. (1992) J. Biol.Chem. 267:6893-6898. See also Moubatin, described in Waxman and Connolly(1993) J. Biol. Chem. 268:5445-5449.

Yet another type of collagen-induced platelet aggregation is isolatablefrom insects as described in the European Publication EP 0 530 937.These proteins are designated "Pallidipins".

Alkaline phosphatase (APase) is an E. coli protein which is secretedinto the periplasmic space. APase is synthesized as a precursor protein,have a 21-amino acid long leader sequence which is clipped off by theleader peptidase in the course of translocation across the bacterialinner membrane into the periplasmic space (Y. Kikuchi et al. (1981)Nucl. Acids Res. 9:5671-5678). Its biosynthesis is regulated by thephosphate concentration of the culture medium, and export of theproducts of heterologous genes placed downstream of the APase promoteris achieved by using low phosphate concentrations (C. Monteilhet et al.(1993) Gene 125:223-228).

The natural source of Pallidipin protein is limited. Processes usingbiotechnological methods are a logical solution for manufacturing ofPallidipin. The expression of Pallidipin in baby hamster kidney cells(EP 0 530 937) occurs at a rate of production which should be increasedin order to express Pallidipin in industrial amounts. Therefore, animproved system of expression was necessary.

Thus, there was a need for an improved process for the manufacture ofrecombinant Pallidipin, which inhibits collagen-induced plateletaggregation, and which process has a high yield and allows reproducibleisolation of the protein with a high degree of purification. The newprocess should not negatively affect the biological activity of theresulting Pallidipin protein.

It has now been found that the problem can be solved by a process ofmanufacture of a recombinant Pallidipin protein (Asp-Pallidipin),wherein the Asp-Pallidipin inhibits the collagen-induced plateletaggregation of mammalian platelets, and wherein the Asp-Pallidipincomprises:

(i) a protein (Pallidipin) selected from the group of Pallidipinproteins, and

(ii) the amino acid aspartic acid,

wherein the aspartic acid is connected by a peptide bond with theN-terminal end of Pallidipin;

whereby the Asp-Pallidipin has the following amino acid sequences:

a) the sequences indicated in

aa) SEQ ID NO:1;

bb) SEQ ID NO:2; or

cc) SEQ ID NO:3; or

b) allelic variants or modifications, or muteins of the sequences in anyof the SEQ ID NOS:1 to 3, which allelic variations or modifications ormuteins do not substantially affect the activity of the protein, or

c) a protein according to any of the SEQ ID NOS:1 to 3 or their variantsor muteins mentioned under b) having post-translational modificationswhich do not substantially affect the activity of the mature protein;

comprising the steps:

aa) transfecting at least one bacterium with an appropriate vector,wherein the vector comprises:

(i) a DNA or cDNA coding for the recombinant Asp-Pallidipin

(ii) a suitable signal peptide sequence which signal sequence is cleavedso that the amino acid aspartic acid is in the position +1 of the aminoacid sequence seen from the position of the Pallidipin and

(iii) a suitable promoter;

bb) expressing the preprotein comprising Asp-Pallidipin and the signalsequence;

cc) transporting the Asp-Pallidipin from the cytoplasm of the bacteriumto the periplasm, cleavage of the preprotein by at least one proteaseduring the transport, producing the,Asp-Pallidipin,

dd) isolating the Asp-Pallidipin by extracting the periplasm, and

ee) purifying the Asp-Pallidipin.

E. coli is a rapid expression system for the production of heterologous,e.g., eukaryotic, proteins. The incorrect folding of proteins is often aproblem during expression of eukaryotic gene products by E. coli, whichcan result in decreased activity of the expression products. Theprobability of correct folding of the protein is much higher when theprotein is transported into the periplasm of the E. coli cells than whenthe protein is maintained in the cytoplasm. Transport of the proteins tothe periplasm is induced by signal peptide sequences attached to themature proteins; these signal peptide sequences together with the matureprotein sequences are designated "preproteins". Correct cleavage of thepreprotein between the signal peptide sequence and the mature protein isnecessary for expression of mature eukaryotic proteins in E. coli ; thesequence of the signal sequence and the sequence of the mature proteinhave an influence on the correct cleavage. Therefore, not all signalpeptide sequences are compatible with all coding sequences.

The E. coli alkaline phosphatase signal sequence is known to beeffective for the export of preproteins. But it is known that thecombination of a given signal peptide sequence and a sequence of amature protein will not necessarily result in good processing of thepreprotein.

Surprisingly, it has been found that the yield of the inventive processof manufacture is 15 times higher than the yield of the expressionsystem using the baby hamster kidney cells. These results are shown inthe Examples. The function and activity of the Asp-Pallidipin, ascompared with the eukaryotic hamster kidney expression system, is notnegatively affected by the inventive process. A further advantage isthat the protease (e.g., leader peptidase) necessary for the cleavage ofthe preprotein is produced by E. coil itself.

Purification of the mature Asp-Pallidipin is much easier using theinventive process than is purification of expressed proteins producedand stored within the cytoplasm of the bacterium. Osmotic shock issufficient to release the Asp-Pallidipin accumulated in the periplasm.

In a preferred embodiment, the DNA encoding the signal peptide sequencecodes for the signal sequence of alkaline phosphatase (APase),preferably E. coli APase.

The invention comprises a process wherein the vector for theAsp-Pallidipin of the invention is derived from the vector pSB94 (U.Boidol et al. (1982) Mol. Gen. Genet. 185:510-512).

A further aspect of the invention is a vector as mentioned before andadditionally a suitable signal peptide, a suitable promoter and, if needbe, a suitable enhancer. Vectors are described in detail in theliterature of the Examples and also in the European publications EP 0480 651; 0 462 632 and 0 173 177.

The bacterium E. coli is the preferred host. Other microorganisms arealso suitable, e.g., Bacillus subtilis.

In addition, the invention comprises a recombinant proteinAsp-Pallidipin, wherein the Asp-Pallidipin inhibits collagen-inducedplatelet aggregation of mammalian platelets, and wherein theAsp-Pallidipin comprises

(i) a protein (Pallidipin) selected from the group of Pallidipinproteins, and

(ii) the amino acid aspartic acid,

wherein the aspartic acid is connected by a peptide bond with theN-terminal end of the Pallidipin;

whereby the Asp-Pallidipin has an amino acid sequences selected from:

a) a sequence selected from

aa) SEQ ID NO:1;

bb) SEQ ID NO:2; or

cc) SEQ ID NO:3; or

b) an allelic variant or modification, or mutein of a sequence of SEQ IDNOS:1 to 3, which allelic variant or modification or mutein hassubstantially the same activity as the Asp-Pallidipin of SEQ ID NO:1 to3; or

c) a protein according to SEQ ID NOS:1 to 3 or their variants or muteinsmentioned under b) having post-translational modifications which do notsubstantially affect the activity of the mature protein.

The invention also comprises a recombinant protein Asp-Pallidipin,wherein the Asp-Pallidipin inhibits collagen-induced plateletaggregation of mammalian platelets, and wherein the Asp-Pallidipincomprises:

(i) a protein (Pallidipin) selected from the group of Pallidipinproteins, and

(ii) the amino acid aspartic acid,

wherein the aspartic acid is connected by a peptide bond with theN-terminal end of the Pallidipin;

whereby the Asp-Pallidipin has the following amino acid sequences:

a) the sequences indicated in

aa) SEQ ID NO:1;

bb) SEQ ID NO:2; or

cc) SEQ ID NO:3; or

b) allelic variants or muteins of the sequences in any of the SEQ IDNOS:1 to 3, which allelic variants or muteins do not substantiallyaffect the activity of the protein, or

c) a protein according to any of the SEQ ID NOS:1 to 3 or their variantsor muteins mentioned under b) having post-translational modificationswhich do not substantially affect the activity of the mature protein;

wherein the Asp-Pallidipin is produced by a process comprising the stepsof:

aa) transfecting at least one bacterium with an appropriate vector,

wherein the vector comprises an operable linkage of:

(i) a first DNA or cDNA molecule, encoding recombinant Asp-Pallidipin,

(ii) a second DNA molecule, encoding a suitable signal peptide sequence,and

(iii) a suitable promoter;

whereby, upon expression, the preprotein comprising the signal peptideand Asp-Pallidipin is cleaved so that the amino acid aspartic acid is inthe position +1 of the amino acid sequence of the mature Asp-Pallidipin,

bb) expressing the preprotein comprising the Asp-Pallidipin and thesignal peptide sequence;

cc) transporting the Asp-Pallidipin from the cytoplasm of the bacteriumto the periplasm, whereby cleavage of the preprotein by at least oneprotease during transport produces the mature Asp-Pallidipin,

dd) isolating the Asp-Pallidipin by extracting the periplasm, and

ee) purifying the Asp-Pallidipin.

In a further preferred embodiment, the Asp-Pallidipin is produced by aprocess comprising the steps of:

culturing a bacterium transfected with an appropriate vector, whereinthe vector comprises an operable linkage of:

(i) a first DNA or cDNA molecule, encoding a recombinant Asp-Pallidipin,

(ii) a second DNA molecule, encoding a suitable signal peptide sequence,and

(iii) a suitable promoter;

whereby, upon expression, the preprotein comprising the signal peptideand Asp-Pallidipin is cleaved so that the amino acid aspartic acid is inthe position +1 of the amino acid sequence of the mature Asp-Pallidipin,under conditions whereby

the preprotein comprising the Asp-Pallidipin and the signal peptidesequence is expressed, and

the Asp-Pallidipin is transported from the cytoplasm of the bacterium tothe periplasm, whereby cleavage of the preprotein by at least oneprotease during transport produces the mature Asp-Pallidipin, andpurifying the Asp-Pallidipin from the periplasm.

Preferred is a protein wherein the signal peptide sequence is the signalsequence of alkaline phosphatase (APase), preferably E. coli APase.

The industrial application of the proteins of the invention is the useof the proteins as a pharmaceutical composition comprising a proteinaccording to the invention in association with a pharmaceuticallyacceptable diluent or carrier.

Allelic variations or modifications as mentioned before includealteration in the sequence of the nucleotides or amino acids, alterationof the genotype or phenotype. At least one nucleotide or one amino acidcan be substituted, deleted or inserted.

Most deletions, insertions and substitutions in particular, are notexpected to produce radical changes in the characteristics of theprotein of the invention. Modified or mutated proteins according to theinvention can be routinely made and screened in order to determine theexact effect of the substitution, deletion, or insertion, by comparisonof the functions of the modified or mutated protein with thecharacteristic functions of the protein of the invention, e.g., theproteins of SEQ ID NOS:1 to 3, or with the native Pallidipin, therebydetermining whether the altered protein has comparable activity, e.g.,biological activity.

The genetic code is degenerative; that is, most amino acids are encodedby more than one codon of three nucleotides. Accordingly, allelicvariation or modification in the nucleotide sequence may or may notalter the amino acid sequence. Therefore, allelic variations areprimarily on the DNA level and may also exist secondarily on the levelof the amino acid sequence.

The DNA sequence coding for the protein of the invention can be modifiedby conventional techniques to produce variations in the final protein ofthe invention which still has substantially the same activity as theprotein of the invention, e.g., the Asp-Pallidipin of SEQ ID NOS:1 to 3,or as compared with the native Pallidipin protein. The activity ismeasured according to the Examples. Thus, one or more amino acids, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. . . up to 15 amino acids, can beadded, substituted or removed without substantially affecting theactivity of the protein of the invention. Substitutions can generally bemade in accordance with the following Table 1 when it is desired tomodulate finely the amino acid sequence of the protein of the invention.

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table1, i.e. selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule, or (c)the bulk of the side chains.

                  TABLE 1    ______________________________________    NORMAL SUBSTITUTIONS OF AMINO ACIDS IN A PROTEIN    ORIGINAL           EXEMPLARY    RESIDUES           SUBSTITUTIONS    ______________________________________    --    Ala                Gly, Ser    Arg                Lys    Asn                Gln, His    Asp                Glu    Cys                Ser    Gln                Asn    Glu                Asp    Gly                Ala, Pro    His                Asn, Gln    Ile                Leu, Val    Leu                Ile, Val    Lys                Arg, Gln, Glu    Met                Leu, Tyr, Ile    Phe                Met, Leu, Tyr    Ser                Thr    Thr                Ser    Trp                Tyr    Tyr                Trp, Phe    Val                Ile, Leu    ______________________________________

Muteins are defined by homology between two compared proteins. Theexpression "homology" comprises similarities of the amino acids and gapsin the sequences of both compared sequences. Similarity of amino acidsis defined for example in Table 1. Preferably, the muteins of thisinvention comprise a sequence of amino acids having a homology of atleast 60%, more preferred at least 80%, much more preferred at least90%, and most preferred at least 95% with the sequence of one of theproteins of SEQ ID NOS:1 to 3.

By "post-translational variations" as mentioned above is meantvariations during or after translation, such as formation of disulfidebridges and chemical modifications of amino acids.

Proteins often form covalent intrachain bonds. These disulfide bonds areformed between cysteine-SH amino acids in the folded protein or in theprotein which folds during translation. The bonds stabilize thethree-dimensional structure of the protein. Such disulfide bonds arerarely formed in protein molecules that are still in the cell cytosolbecause the high intracellular concentration of the --SS--(disulfide)reducing agent glutathione breaks most of such bonds. Once the proteinsare outside the cytoplasm, are secreted or are on the cell surface, theyoften form additional covalent intrachain bonds.

Furthermore, the amino acids may be altered as described in PCTApplication WO 91/10684. Other alterations of the side chains of theamino acids are possible.

The protein of the invention has at least a purity of 40%, preferably atleast 60%, more preferably at least 80% and most preferably at least90%. The purity is defined by the amount of the protein of the inventionin relation to the total amount of protein. Using the purificationmethods described in the Examples, no other proteins other than theproteins of the invention are detectable.

Using the purified protein of the invention, monoclonal antibodies canbe produced according to the well-known Koehler and Milstein methodwhich, in particular, comprises conventionally immunizing mice orrabbits with the purified protein of the invention as immunogen,followed by the production of hybridomas from antibody-producing cellsof the mouse or rabbit.

The preferred embodiment of the invention is the protein mentioned inSEQ ID NO:1, expressed in E. coli strain E 15.

The Asp-Pallidipin exhibits pharmacological activity and may, therefore,be useful as a pharmaceutical. Asp-Pallidipin can be used in apharmaceutical composition comprising Asp-Pallidipin in association witha pharmaceutically acceptable diluent or carrier. Additionally, theinvention comprises a pharmaceutical composition comprising apharmaceutically active Asp-Pallidipin according to the invention and apharmaceutically acceptable salt or a pharmaceutically acceptablecarrier.

In particular, Asp-Pallidipin inhibits collagen-induced plateletaggregation and inhibits adhesion of tumor cells, preferably ofmetastatic tumor cells, to collagen.

Asp-Pallidipin inhibits platelet aggregation. The test system isdescribed in the Examples. Asp-Pallidipin significantly inhibitsplatelet aggregation at a concentration of 0.5 to 50 μg protein. Themost preferred Asp-Pallidipin, the protein of SEQ ID NO:1, has an IC₅₀of 50 nmol/L of highly purified protein according to the Examples.Asp-Pallidipin inhibits platelet aggregation at concentrations of from 5nmol/L to about 1,000 nmol/L.

The results from the in vitro test systems indicate that the proteins ofthe invention can be used as a medicament or can be used for medicaltreatment. The test results for the in vitro system can be correlatedwith the in vivo system, because it is an established system in thisfield. R. J. Shebuski et al. (1990) Thrombosis and Haemostasis,64:576-581.

Asp-Pallidipin can be administered by intraperitoneal injections, whichcan be given daily or at 2 to 3 times a week. When animals receive dailyinjections to achieve a blood concentration of 100 nmol/L, theirplatelet aggregation is reduced. No serious side effect are monitoredunder these conditions. Asp-Pallidipin causes this inhibition ofplatelet aggregation in mice at daily dosages which achieve a bloodconcentration of from about 10 nmol/L to 1,000 nmol/L.

Asp-Pallidipin is, therefore, useful for the treatment ofatherosclerotic or thrombotic disease lesions or for preventingreocclusion after treatment of myocardial infarction. Asp-Pallidipin canbe used as an antiatherosclerotic and antithrombotic agent in mammals,including humans, e.g., to treat atherosclerotic/thrombotic lesions, forexample due to rupture of atherosclerotic plaques or those due toperturbation or removal of endothelium, e.g., in sepsis or transplants,or to treat unstable angina. It can also be used to prevent reocclusionafter treatment of myocardial infarction by fibrinolysis or byangioplasty (PTCA). If fibrinolytic therapy (with streptokinase, t-PA orother plasminogen activators) is applied to treat myocardial infarction,Asp-Pallidipin can be used as an adjuvant agent to prevent reocclusionof the blood vessel. Treatment of myocardial infarction with a ballooncatheter (PTCA) also injures the vessel wall and this may lead toformation of a new thrombus. This can be prevented by administeringAsp-Pallidipin during and after the procedure. Asp-Pallidipin can beused in coronary angioplasty as well as in other angioplastyapplications.

The invention provides

a) the use of a protein of the invention for manufacture of a medicamentfor treatment of atherosclerotic or thrombotic disease or for preventingreocclusion after treatment of myocardial infarction (thus, the proteinsare useful as prophylactically effective medicaments for treatment ofpatients known to be at risk of developing a disease or condition);

b) a method of treatment of atherosclerotic or thrombotic disease or forpreventing reocclusion after treatment of myocardial infarction, whichcomprises administration of a disease-suppressing effective amount ofthe protein of the invention to a patient in need of such treatment;

c) a pharmaceutical composition for treatment of atherosclerotic orthrombotic disease or for preventing reocclusion after treatment ofmyocardial infarction which comprises a protein of the invention and apharmaceutically acceptable carrier or diluent.

For these indications the appropriate dosage will, of course, varydepending upon, for example, the compound of the invention employed, thehost, the mode of administration and the nature and severity of thecondition being treated. However, in general, satisfactory results inanimals are indicated to be obtained at daily dosages to achieve a bloodconcentration of from 10 to 1,000 nmol/L, preferably at daily dosages offrom 30 to 300 nmol/L.

The proteins of the invention may be administered by any conventionalroute, in particular enterally or parenterally, e.g. in the form ofinjectable solutions or suspensions. Intraperitoneal injection ispreferred.

The protein of the SEQ ID NO:1 is the preferred compound.

The present invention provides pharmaceutical compositions comprisingcompounds of the invention in association with at least onepharmaceutical carrier or diluent. Such compositions may be manufacturedin conventional manner. See Remington's Pharmaceutical Science, 15^(th)ed. Mack Publishing Company, Easton Pa. (1980).

The proteins of the invention also inhibit adhesion of metastatic tumorcells to collagen. The test system is described in the Examples. Theproteins of the invention show a significant adhesion-inhibition ofmetastatic tumor cells to collagen in a concentration of 1 to 100 μgprotein.

The test of the most preferred protein, the protein of SEQ ID NO:1,shows a value of the IC₅₀ of 100 nmol/L of the highly purified proteinaccording to the Examples 2 and 15. The proteins of the invention showthe adhesion-inhibition of metastatic tumor cells to collagen atconcentration of from 10 to 2,000 nmol/L.

The results from the in vitro test systems indicate that the proteins ofthe invention can be used as a medicament or can be used for medicaltreatment. The test results can be transferred from the in vitro systemto the in vivo system, because it is an established system in thisfield. Chan et al. (1990), Science, 2:1600-1602.

The proteins of the invention can be administered during and aftersurgical operations of the primary tumor to prevent formation ofmetastasis by detached tumor cells which may enter the blood streamduring operation. These anti-metastatic effects can be demonstrated inan "experimental" and "spontaneous" animal model as described by Chan etal. (1990), Science, 2:1600-1602.

The proteins of the invention can be administered by intraperitonealinjections which are given daily or at 2 to 3 times a week. When animalsreceive daily injections to achieve a blood concentration of 200 nmol/L,they have a reduced adhesion of metastatic tumor cells measured bycounting the value of centers of settled metastatic cells. No seriousside effects are noted under these conditions.

The proteins of the invention show this adhesion-inhibition ofmetastatic tumor cells to collagen in mice at daily dosages to achieve ablood concentration of from 20 to 2,000 nmol/L, preferablyconcentrations of from 60 to 600 nmol/L.

The proteins of the invention are, therefore, useful for the treatmentof cancer; especially cancer with metastatic tumor cells, mostpreferably cancer with highly metastatic tumor cells.

The invention thus provides

a) the use of a protein of the invention for manufacture of a medicamentfor treatment of cancer with metastatic tumor cells (the proteins arethus useful for prophylactically effective medicaments administeredbefore, e.g., surgical removal of tumors);

b) a method of treatment of cancer with metastatic tumor cells, whichcomprises administration of a disease-suppressing effective amount ofthe protein of the invention to a patient in need of such treatment;

c) a pharmaceutical composition for treatment of cancer with metastatictumor cells which comprises a protein of the invention and apharmaceutically acceptable carrier or diluent.

For these indications the appropriate dosage will, of course, varydepending upon, for example, the compound of the invention employed, thehost, the mode of administration and the nature and severity of thecondition being treated. However, in general, satisfactory results inanimals are indicated to be obtained at daily dosages to achieve a bloodconcentration of from 20 to 2,000 nmol/L, preferably at daily dosages of60 to 600 nmol/L.

The proteins of the invention may be administered by any conventionalroute, in particular enterally or parenterally, e.g. in the form ofinjectable solutions or suspensions.

The protein of the SEQ ID NO:1 is the preferred compound.

The present invention provides pharmaceutical compositions comprisingcompounds of the invention in association with at least onepharmaceutical carrier or diluent. Such compositions may be manufacturedin conventional manner. See Remington's Pharmaceutical Science, 15^(th)ed. Mack Publishing Company, Easton, Pa. (1980).

In another aspect of this invention, there is provided DNA sequences,vectors containing these sequences, cells containing said vectors,methods of recombinantly producing proteins and antibodies to theproteins of this invention. Also provided are isolated and/orrecombinant DNA sequences (e.g., genomic or cDNA) coding for a proteinwhich inhibits collagen-induced aggregation of human platelets. In astill further aspect, the invention provides recombinantly-producedproteins of this invention, e.g., having the sequences disclosed herein.

By the term "isolated" is meant that the inhibitor of this invention orother entity is present in a form separated from (purified from)components with which it is produced recombinantly or synthetically. Alldegrees of such isolation or purification are included generically.Preferred are degrees of isolation or purification whereby the inhibitoris useful for pharmaceutical purposes. For example, such degrees ofisolation (e.g., activities or purities) can be routinely achieved bychromatographic techniques such as those used in the examples. Furtherpurifications, e.g., to homogeneity, can be routinely achieved usingconventional methods, such as those described in the following texts:

Methods of Enzymology, Volume 182, Guide to Protein Purification, ed.Murray P. Deutscher, Academic Press 1990;

Protein Purification Applications--A Practical Approach. ed. E.L.V.Harris and S. Angel, IRL-Press 1990;

Protein Purification, Principles and Practice, Robert Scopes,Springer-Verlag 1982; and

Protein Purification, Principles, High Resolution Methods andApplications, ed. J.-C. Janson and L. Ryden, VCH publishers 1989.

Purity can be determined by any one of a number of routine methods,e.g., SDS polyacrylamide gel electrophoresis, analytical HPLC, etc.Purified inhibitor can be used to determine the amino acid sequence ofthe protein according to methods fully routine to one of ordinary skillin the art. Hewick, R. M. et al. (1981) J. Biol. Chem. 256, 7990-7997.

The amino acid sequence of the inhibitor of the present invention can beused to determine the sequence of suitable DNA probes, which can be usedfor finding new inhibitors, e.g., in other species. Such probes can beroutinely synthesized, e.g., using automated DNA synthesizers, andscreening of genomic or cDNA libraries is similarly routine for one ofordinary skill in the art. (See International Publication WO 90/07861,dated 26 Jul., 1990)

Therefore, the present invention also includes the DNA sequencecorresponding to (coding for) both the DNA sequence (gene) for theAsp-Pallidipin, when isolated from the natural environment, e.g., insolution or on a vector, as well as muteins thereof. Methods forproducing muteins are also routine and conventional for one of ordinaryskill in the art, as are screening methods for testing the efficacy ofsuch new proteins, e.g., as described herein.

Suitable muteins are those having at least a fraction, e.g., at least5%, preferably at least 50%, most preferably at least 90% of thebiological activity, e.g., collagen-induced platelet aggregationinhibition, of inhibitor as described herein.

Further the invention comprises a method of purification, wherein thepurifying comprises the following consecutive steps:

(i) purifying by cation exchange chromatography;

(ii) purifying by anion exchange and

(iii) purifying by size exclusion.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative and not limitative ofthe remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius; and, unless otherwise indicated,all parts and percentages are by weight.

The entire disclosures of all applications, patents and publications,cited above and below, if any, including EP 94250224.6, filed Sep. 2,1994, are hereby incorporated by reference.

EXAMPLES Example 1

Construction of the Expressing Vector

For construction of the inventive vector, coding for the Asp-Pallidipin,the following sense and antisense primers are employed: ##STR1## areused.

PCR is for 8 cycles of 2 minutes at 94° C., 1 minute and 30 seconds at42° C. and 2 minutes and 30 seconds at 72° C. using p3 and p4, as primerpairs and 1 μg template DNA. After gel purification and digestion withNrul and BamHI, the fragment is subcloned into pSB/pho. The plasmid isprepared by Xmal digestion followed by Mung bean treatment to blunt the5' overhang and BamHI digestion.

The construct is checked by complete DNA sequencing of the insertedfragments using the dideoxy chain-termination method (F. Sanger et al.(1977) Proc. Nat'l. Acad. Sci. USA 74:5463-5467) and a sequencing kitwith ³⁵ S!dATP. Transformation of competent E. coli E15 with Pallidipinexpression construct or empty plasmid (mock transformation) is carriedout using standard methods (J. Sambrook et al. (1989) Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY).

Example 2

Expression and Extraction of Asp-Pallidipin

For the pSB/pho plasmid (derived from pSB94; see J. Daum et al. (1989)Eur. J. Biochem. 185:347-354), overnight E15 cultures (S. J. Hayashi etal. (1964) J. Biol. Chem. 239:3091-3106) are diluted to 8% (v/v) in lowphosphate medium and grown for 6 hours at 37° C. (A. Becker et al.(1994) Protein Expression and Purification 5:50-56). The bacteria areharvested after induction by centrifugation and resuspended in 1/10volume of 50 mmol/L Tris-HCl (pH 8.0)/100 mmol/L NaCI. This preparationis frozen, thawed, and centrifuged, thus giving a first supernatantfraction (SN1). The bacterial pellet is resuspended and equilibrated for10 min at room temperature in 0.5 mol/L saccharose beforecentrifugation. The supernatant (SN2) is kept for analysis, while thepellet is resuspended in ice-cold water supplemented with 1 mmol/L PMSF(phenylmethylsulfonylfluoride) and incubated for 10 min on ice.Following this osmotic shock, the cells are centrifuged and thesupernatant (SN3) is collected. The pellet containing the cytoplasmicfraction (CF) is resuspended in 50 mmol/L Tris-HCI (pH 8.0)/100 mmol/LNaCl.

Example 3

Purification of Recombinant Asp-Pallidipin

The supernatant fraction containing the bulk of recombinantAsp-Pallidipin (SN1) is adjusted to pH 4.0 with acetic acid and appliedto a cation exchange column (Mono S, Pharmacia) using an FPLC system.After washing with a gradient of 0 to 500 mmol/L NaCI in sodium acetatepH 4.0, elution of Asp-Pallidipin is achieved with 20 mmol/L NaPi, pH7.0. Eluted fractions containing Asp-Pallidipin, as judged bySDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting, arepooled, adjusted to pH 8.0 and applied to an anion exchange column(Fractogel-EMD-TMAE 650, Merck). Elution of Asp-Pallidipin is achievedwith a gradient of 0 to 1 mol/L NaCl in 20 mmol/L sodium acetate pH 8.4.For the final purification, eluent fractions containing Asp-Pallidipinare pooled, concentrated in the Seed-Vac (Bachofer) and subjected tosize exclusion chromatography using Superose 12 (Pharmacia).

Example 4

Platelet Aggregation Assay

The assay is carried out essentially as described in the publication ofC. Noeske-Jungblut (C. Noeske-Jungblut (1994) J. Biol. Chem.269:5050-5053). Briefly, human blood is collected into 1/6 volume 71mmol/L citric acid/85 mmol/L trisodium citrate/111 mmol/L glucose.Platelet-rich plasma is obtained by centrifugation at 135 g for 20 min.Asp-Pallidipin is incubated with 500 μl platelet-rich plasma for 1 minat 37° C. before the addition of collagen (2 μg/ml). The aggregation ismonitored using a Micron aggregometer and the maximum value isdetermined. The IC₅₀ has a value of about 50 nM. A significantdifference between the compounds with or without Asp in the firstposition cannot be seen.

The biological activity and the yield of Asp-Pallidipin purified fromthe periplasmic space of E. coli is determined. Platelet-rich plasma isincubated with Asp-Pallidipin and controls. Aggregation is induced byadding collagen. Wild-type Pallidipin purified from saliva is used as apositive control. Further some other constructs are used as controls,showing the advantage of the inventive Asp-Pallidipin. The protein ofthe invention and the controls show different yields (Table 2).

                  TABLE 2    ______________________________________    Strain               Yield    ______________________________________    recombinant Pallidipin                         461 μg    recombinant arginyl-Pallidipin                         298 μg    recombinant aspartyl-Pallidipin                         864 μg    ______________________________________

Example 5

The adhesion of tumor cells to collagen is decreased in the presence ofthe protein of the invention

The protein of the invention inhibits the adhesion of tumor cells to acollagen matrix. Therefore, migrating tumor cells can be preventedpartially or completely from settling down in organs or blood vessels,when the protein of the invention is within the blood or the plasma ofthe patient.

MTLn3 cells (rat mammary tumor cells) are labelled with ⁵¹ Cr. A wellplate is coated with collagen (type III) at 4° C. overnight. 2·10⁴labelled cells in 500 μl DMEM F12 medium, 20 mmol/L Hepes, 1 mmol/Lbicarbonate, 1% BSA are first incubated with 0,2,5 or 10 μl protein ofthe invention ("Superose Pool", 0.5 mg protein/ml) respectively for 10min at 37° C. Then this suspension is transferred to a collagen-coatedwell and incubated for 2 h at 37° C. Thereafter, the wells are washedand the adherent cells are removed with 1 mol/L NaOH. The radioactivityof the adherent cells is counted.

                  TABLE 3    ______________________________________    amount of the inhibitor added                       cell attachment    μl              (cpm)    ______________________________________    0                  2215    2                  2071    5                  1608    10                 1081    ______________________________________

Example 6

Antibody production

About 100 μg of the inhibitor purified according to the examples areadded to 0.5 ml of complete Freund's adjuvant and the emulsion isinjected s.c. into a rabbit. After 2 weeks a second injection is givenconsisting of about 80 μg purified inhibitor and 0.5 ml incompleteFreund's adjuvant. After the injection, several samples of serum aretaken to check the production of specific antibodies. They are assayedin a Western blot. 20 ng of the purified inhibitor is applied on a 12.5%SDS-polyacrylamide gel and the electrophoresis, blotting and detectionare done according to standard methods described by E. Harlowe, D. Lane,(1988) Antibodies: a laboratory manual, Cold Spring Harbor Laboratory(dilution of the test serum 1:500, goat anti-rabbit peroxidaseconjugated IgG as second antibody, detection with the ECL-kit fromAmersham International, Amersham, UK). The blot shows that the antiserumspecifically reacts with the purified inhibitor.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 5    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 172 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    AspGluGluCysGluLeuMetProProGlyAspAsnPheAspLeuGlu    151015    LysTyrPheSerIleProHisValTyrValThrHisSerArgAsnGly    202530    ProLysGluGlnValCysArgGluTyrLysThrThrLysAsnSerAsp    354045    GlyThrThrThrThrThrLeuValThrSerAspTyrLysThrGlyGly    505560    LysProTyrHisSerGluLeuLysCysThrAsnThrProLysSerGly    65707580    GlyLysGlyGlnPheSerValGluCysGluValProAsnGlyAsnGly    859095    GlyLysLysLysIleHisValGluThrSerValIleAlaThrAspTyr    100105110    LysAsnTyrAlaLeuLeuGlnSerCysThrLysThrGluSerGlyIle    115120125    AlaAspAspValLeuLeuLeuGlnThrLysLysGluGlyValAspPro    130135140    GlyValThrSerValLeuLysSerValAsnTrpSerLeuAspAspTrp    145150155160    PheSerArgSerLysValAsnCysAspAsnMetLys    165170    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 171 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    AspGluGluCysGluLeuMetProProGlyAspAsnPheAspLeuGlu    151015    LysTyrPheSerIleProHisValTyrValThrHisSerArgAsnGly    202530    ProLysGluGlnValCysArgGluTyrLysThrThrLysAsnSerAsp    354045    GlyThrThrThrThrLeuValThrSerAspTyrLysThrGlyGlyLys    505560    ProTyrHisSerGluLeuLysCysThrAsnThrProLysSerGlyVal    65707580    LysGlyGlnPheSerValGluCysGluValProAsnGlyAsnGlyGly    859095    LysLysLysIleHisValGluThrSerValIleAlaThrAspTyrLys    100105110    AsnTyrAlaLeuLeuGlnSerCysThrLysThrGluSerGlyIleAla    115120125    AspAspValLeuLeuLeuGlnThrLysLysGluGlyValAspProGly    130135140    ValThrSerValLeuLysSerValAsnTrpSerLeuAspAspTrpPhe    145150155160    SerArgSerLysValAsnCysAspAsnMetLys    165170    (2) INFORMATION FOR SEQ ID NO: 3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 172 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    AspGluGluCysGluLeuMetProProGlyAspAsnPheAspLeuGlu    151015    LysTyrPheSerIleProHisValTyrValThrHisSerArgAsnGly    202530    ProLysGluGlnValCysArgGluTyrLysThrThrLysAsnSerAsp    354045    GlyThrThrThrThrThrLeuValThrSerAspTyrLysThrGlyGly    505560    LysProTyrHisSerGluLeuLysCysThrAsnThrGlnLysSerGly    65707580    GlyLysGlyGlnPheSerValGluCysGluValProAsnGlyAsnGly    859095    GlyLysLysLysIleHisValGluThrSerValIleAlaThrAspTyr    100105110    LysAsnTyrAlaLeuLeuGlnSerCysThrLysThrGluSerGlyIle    115120125    AlaAspAspValLeuLeuLeuGlnThrLysLysGluGlyValAspPro    130135140    GlyValThrSerValLeuLysSerValAsnTrpSerLeuAspAspTrp    145150155160    PheSerArgSerLysValAsnCysAspAsnMetLys    165170    (2) INFORMATION FOR SEQ ID NO: 4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "sense primer"    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    GCGATATCGCGACGAAGAATGCGAACTCATG31    (2) INFORMATION FOR SEQ ID NO: 5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "antisense primer"    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:    GCGATAGGATCCAAGCTTATTACTTCATGTTATC34    __________________________________________________________________________

We claim:
 1. A method of producing a recombinant Pallidipin protein (Asp-Pallidipin), wherein the Asp-Pallidipin inhibits collagen-induced platelet aggregation of mammalian platelets, and wherein the Asp-Pallidipin comprises:(i) a protein (Pallidipin) selected from the group of Pallidipin proteins, and (ii) the amino acid aspartic acid, wherein the aspartic acid is connected by a peptide bond with the N-terminal end of the Pallidipin;whereby the Asp-Pallidipin has an amino acid sequence selected from: a) the sequences indicated inaa) SEQ ID NO:1; bb) SEQ ID NO:2; or cc) SEQ ID NO:3; or b) an allelic variant of the sequences of any of the SEQ ID NOS:1 to 3, or c) a protein according to any of the SEQ ID NOS: 1 to 3 or a variant mentioned under b), having a post-translational modification which does not substantially affect the platelet aggregation inhibitory activity of the mature protein;comprising the steps of: aa) transfecting at least one bacterium with an appropriate vector, wherein the vector comprises an operable linkage of:(i) a first DNA or cDNA molecule, encoding recombinant Asp-Pallidipin, (ii) a second DNA molecule, encoding a suitable signal peptide sequence, and (iii) a suitable promoter; whereby, upon expression, the preprotein comprising the signal peptide and Asp-Pallidipin is cleaved so that the amino acid aspartic acid is in the position +1 of the amino acid sequence of the mature Asp-Pallidipin, bb) expressing the preprotein comprising the Asp-Pallidipin and the signal peptide sequence; cc) transporting the Asp-Pallidipin from the cytoplasm of the bacterium to the periplasm, whereby cleavage of the preprotein by at least one protease during transport produces the mature Asp-Pallidipin, dd) isolating the Asp-Pallidipin by extracting the periplasm, and ee) purifying the Asp-Pallidipin.
 2. A process according to claim 1, wherein the DNA encoding the signal sequence codes for the signal sequence of alkaline phosphatase (APase).
 3. A process according to claim 1, wherein the bacterium is E coli.
 4. A method of treatment of atherosclerotic or thrombotic disease or for preventing reocclusion after treatment of myocardial infarction, comprising administering to a patient in need of such treatment an effective amount of an Asp-Pallidipin produced by a method of claim
 1. 5. A method of treatment of cancer with metastatic tumor cells, comprising administering to a patient in need of such treatment an effective amount of Asp-Pallidipin produced by a method of claim
 1. 6. A process of purification of an Asp-Pallidipin produced according to claim 1, wherein the purifying comprises the following consecutive steps:(i) purifying by cation exchange chromatography; (ii) purifying by anion exchange; and (iii) purifying by size exclusion.
 7. A method of producing a recombinant Pallidipin protein (Asp-Pallidipin), wherein the Asp-Pallidipin inhibits the collagen-induced platelet aggregation of mammalian platelets, and wherein the Asp-Pallidipin comprises:(i) a protein (Pallidipin) selected from the group of Pallidipin proteins, and (ii) the amino acid aspartic acid, wherein the aspartic acid is connected by a peptide bond with the N-terminal end of Pallidipin;whereby the Asp-Pallidipin has an amino acid sequence selected from: a) the sequences indicated inaa) SEQ ID NO:1; bb) SEQ ID NO:2; or cc) SEQ ID NO:3; or b) an allelic variant of the sequences in any of the SEQ ID NOS: 1 to 3, or c) a protein according to any of the SEQ ID NOS: 1 to 3 or a variant mentioned under b), having a post-translational modification which does not substantially affect the platelet aggregation inhibitory activity of the mature protein;comprising: culturing a bacterium transfected with an appropriate vector, wherein the vector comprises an operable linkage of:(i) a first DNA or cDNA molecule, encoding a recombinant Asp-Pallidipin, (ii) a second DNA molecule, encoding a suitable signal peptide sequence, and (iii) a suitable promoter; whereby, upon expression, the preprotein comprising the signal peptide and Asp-Pallidipin is cleaved so that the amino acid aspartic acid is in the position +1 of the amino acid sequence of the mature Asp-Pallidipin,under conditions whereby (A) the preprotein comprising the Asp-Pallidipin and the signal peptide sequence is expressed, and (B) the Asp-Pallidipin is transported from the cytoplasm of the bacterium to the periplasm, whereby cleavage of the preprotein by at least one protease during transport produces the mature Asp-Pallidipin, and purifying the thus-produced Asp-Pallidipin from the periplasm.
 8. A recombinant protein Asp-Pallidipin, wherein the Asp-Pallidipin inhibits collagen-induced platelet aggregation of mammalian platelets, and wherein the Asp-Pallidipin comprises:(i) a protein (Pallidipin) selected from the group of Pallidipin proteins, and (ii) the amino acid aspartic acid, wherein the aspartic acid is connected by a peptide bond with the N-terminal end of the Pallidipin;whereby the Asp-Pallidipin has an amino acid sequence selected from: a) the sequences indicated inaa) SEQ ID NO:1; bb) SEQ ID NO:2; or cc) SEQ ID NO:3; or b) an allelic variant of the sequences in any of the SEQ ID NOS: 1 to 3, or c) a protein according to any of the SEQ ID NOS: 1 to 3 or a variant mentioned under b), having a post-translational modification which does not substantially affect the platelet aggregation inhibitory activity of the mature protein.
 9. A pharmaceutical composition comprising an effective amount of an Asp-Pallidipin according to claim 8 in association with a pharmaceutically acceptable diluent or carrier.
 10. A method of treatment of atherosclerotic or thrombotic disease or for preventing reocclusion after treatment of myocardial infarction, comprising administering to a patient in need of such treatment an effective amount of an Asp-Pallidipin of claim
 8. 11. A recombinant protein Asp-Pallidipin, wherein the Asp-Pallidipin inhibits collagen-induced platelet aggregation of mammalian platelets, and wherein the Asp-Pallidipin comprises(i) a protein (Pallidipin) selected from the group of Pallidipin proteins, and (ii) the amino acid aspartic acid, wherein the aspartic acid is connected by a peptide bond with the N-terminal end of the Pallidipin;whereby the Asp-Pallidipin has an amino acid sequencer selected from: a) the sequences indicated inaa) SEQ ID NO:1; bb) SEQ ID NO:2; or cc) SEQ ID NO:3; or b) an allelic variant of the sequences in any of the SEQ ID NOS: 1 to 3, or c) a protein according to any of the SEQ ID NOS: 1 to 3 or a variant mentioned under b), having a post-translational modification which does not substantially affect the platelet aggregation inhibitory activity of the mature protein;wherein the Asp-Pallidipin is produced by a process comprising the steps: aa) transfecting at least one bacterium with an appropriate vector, wherein the vector comprises an operable linkage of:(i) a first DNA or cDNA molecule, encoding recombinant Asp-Pallidipin, (ii) a second DNA molecule, encoding a suitable signal peptide sequence, and (iii) a suitable promoter; whereby, upon expression, the preprotein comprising the signal peptide and Asp-Pallidipin is cleaved so that the amino acid aspartic acid is in the position +1 of the amino acid sequence of the mature Asp-Pallidipin, bb) expressing the preprotein comprising the Asp-Pallidipin and the signal peptide sequence; cc) transporting the Asp-Pallidipin from the cytoplasm of the bacterium to the periplasm, whereby cleavage of the preprotein by at least one protease during transport produces the mature Asp-Pallidipin, dd) isolating the Asp-Pallidipin by extracting the periplasm, and ee) purifying the Asp-Pallidipin.
 12. A Asp-Pallidipin according to claim 11, wherein the DNA encoding the signal peptide sequence encodes a signal sequence of alkaline phosphatase (APase).
 13. A recombinant protein Asp-Pallidipin, wherein the Asp-Pallidipin inhibits collagen-induced platelet aggregation of mammalian platelets, and wherein the Asp-Pallidipin comprises:(i) a protein (Pallidipin) selected from the group of Pallidipin proteins, and (ii) the amino acid aspartic acid, wherein the aspartic acid is connected by a peptide bond with the N-terminal end of Pallidipin;whereby the Asp-Pallidipin has an amino acid sequence selected from: a) the sequences indicated inaa) SEQ ID NO:1; bb) SEQ ID NO:2; or cc) SEQ ID NO:3; or b) an allelic variant of the sequences in any of the SEQ ID NOS: 1 to 3, or c) a protein according to any of the SEQ ID NOS: 1 to 3 or a variant mentioned under b), having a post-translational modification which does not substantially affect the platelet aggregation inhibitory activity of the mature protein;wherein the Asp-Pallidipin is produced by a process comprising: culturing a bacterium transfected with an appropriate vector, wherein the vector comprises an operable linkage of: (i) a first DNA or cDNA molecule, encoding a recombinant Asp-Pallidipin, (ii) a second DNA molecule, encoding a suitable signal peptide sequence, and (iii) a suitable promoter; whereby, upon expression, the preprotein comprising the signal peptide and Asp-Pallidipin is cleaved so that the amino acid aspartic acid is in the position +1 of the amino acid sequence of the mature Asp-Pallidipin,under conditions whereby (A) the preprotein comprising the Asp-Pallidipin and the signal peptide sequence is expressed, and (B) the Asp-Pallidipin is transported from the cytoplasm of the bacterium to the periplasm, whereby cleavage of the preprotein by at least one protease during transport produces the mature Asp-Pallidipin, and purifying the Asp-Pallidipin, from the periplasm.
 14. A recombinant vector comprising an operable linkage of:(i) a first DNA or cDNA molecule, encoding a recombinant Asp-Pallidipin, (ii) a second DNA molecule, encoding a suitable signal peptide sequence, and (iii) a suitable promoter;whereby, upon expression in a suitable bacterial host, the preprotein comprising the signal peptide and Asp-Pallidipin is cleaved so that the amino acid aspartic acid is in the position +1 of the amino acid sequence of the mature Asp-Pallidipin.
 15. A recombinant vector of claim 14, wherein the DNA or cDNA molecule encodes an Asp-Pallidipin having an amino acid sequence selected from:a) the sequences indicated inaa) SEQ ID NO:1; bb) SEQ ID NO:2; or cc) SEQ ID NO:3; or b) an allelic variant of the sequences in any of the SEQ ID NOS: 1 to
 3. 16. A bacterial host transformed with a vector of claim
 14. 17. A bacterial host of claim 16, which is an E coli. 